(1) Field of the Invention
The present invention relates to the field of pesticidal compound analysis. Such methods are useful for monitoring the behaviour of such pesticidal compounds once they have been applied, and such methods are also useful during marketing authority procedures for such pesticidal compounds.
(2) Description of Related Art
The compounds that can be analyzed by means of the method of analysis according to the invention are compounds that are useful for the protection of plants and also some of the compounds that are metabolites of these biologically active compounds. Some of these metabolites can also exhibit biological activities.
Methods for analyzing pesticidal compounds are known. In particular, an analytical method for determining the fosetyl-Al residue and its main metabolite, phosphorous acid, in drinking water or in surface water is known.
Such a known method uses (trimethylsilyl)diazomethane (TMSD) as a derivatizing agent.
Generally, such a method comprises the following steps:                concentration of the water samples;        derivatization with (trimethylsilyl)diazomethane of an aliquot of the concentrated sample (substitution of a hydrogen atom with a methyl group);        purification of the derivatized sample by liquid-liquid partition with dichloromethane.        
The analysis is carried out by gas chromatography on a semi-capillary column by means of a flame photometric detector (or FPD) in the phosphorous mode and the quantification takes external standards as reference. The use of a thermionic detector is also possible.
This known method is carried out according to Scheme 1 below:

With this method, the limits of detection (LODs) are as follows:                for fosetyl-Al:                    0.05 μg/l for drinking water (mineral or mains water);            0.05 μg/l for surface water (river water);                        for phosphorous acid:                    0.7 μg/l for drinking water (mineral or mains water);            2.5 μg/l for surface water (river water).                        
With this method, the limits of quantification (LOQs) are as follows:                for fosetyl-Al:                    0.7 μg/l for drinking water (mineral or mains water);            1 μg/l for surface water (river water);                        for phosphorous acid:                    2.0 μg/l for drinking water (mineral or mains water);            4.0 μg/l for surface water (river water).                        
This known method of analysis has been used for the following substrates: mineral water, mains water and water from the Rhone river. The method of analysis has been validated for various types of water, by analysis of non treated control samples enriched in fosetyl-Al and in phosphorous acid to the limits of quantification and to 10 times these limits.
The analysis of these enriched samples gives recovery rate values for fosetyl-Al or for phosphorous acid, which are compared with the expected theoretical values.
As another known method of analysis, mention may be made of a method for analyzing fosetyl-Al or phosphorous acid residues present in soils taken in Chazay d'Azergues (France), in Goch (Germany) and in Seville (Spain).
This method itself also uses (trimethylsilyl)diazomethane. It is also carried out according to Scheme 1.
In this course of this method, the fosetyl-Al and phosphorous acid residues are extracted from the soil samples by agitation in the presence of an ammonia buffer solution, and then the cations that are present are removed from the extracts by means of an ion exchange resin and the water is evaporated from the samples. Finally, the extracts obtained are derivatized by the action of (trimethylsilyl)diazomethane.
Subsequently, the quantification is carried out by gas chromatography on a semi-capillary column using a flame photometric detector (in the phosphorous mode) with an external standard.
The limit of quantification (LOQ) of this method is 0.100 mg/kg for each of the compounds.
The control samples were enriched in fosetyl-Al or in phosphorous acid up to the limit of quantification and also up to 100 times this limit.
Another known method for analyzing residues concerns the analysis of fosetyl-Al and phosphorous acid residues in plant samples, derived both from fruits and from vegetables.
This method itself also uses (trimethylsilyl)diazomethane. It is also carried out according to Scheme 1.
In the course of this method, the fosetyl-Al and phosphorous acid residues are extracted from the plant samples by milling in a mixture of water and acetonitrile. The extracts are subsequently purified using a C18 cartridge and are then derivatized by the action of (trimethylsilyl)diazomethane.
Subsequently, the quantification is carried out by gas chromatography on a semi-capillary column using a flame photometric detector (in the phosphorous mode) with an external standard.
The limit of quantification of this known method is 0.50 mg/kg for each of the products, with the exception of hop, for which this limit is 2.0 mg/kg for fosetyl-Al and 20.0 mg/kg for phosphorous acid.
This method has been used on samples of bunches of grapes, of oranges, of bananas, of strawberries, of lettuce and of cucumbers. The control samples were enriched, in particular up to the limit of quantification.
Yet another known method for analyzing residues concerns the analysis of fosetyl-Al or phosphorous acid residues in animal tissues or products of animal origin, such as milk, bovine meat, bovine kidneys, bovine liver or eggs.
According to this study, the residues of compounds to be analyzed are extracted from the samples by double milling in a water/acetonitrile mixture (50/50, 20/80 for milk).
An aliquot of the extract is subsequently purified by means of a C18 cartridge (except for milk). The purified extract is subsequently derivatized with a solution of TMSD.
This method of analysis also follows Scheme 1.
The quantification is carried out by gas chromatography on a DB Wax column using a flame photometric detector in the phosphorous mode.
The limits of quantification are as follows:                0.50 mg/kg for fosetyl-Al and phosphorous acid in bovine meat, bovine kidney, bovine liver and eggs;        0.10 mg/kg for fosetyl-Al and phosphorous acid in milk.        
For this method, non treated control samples were prepared and analyzed, along with samples enriched to the limit of quantification and also to several times this limit.
The known methods of analysis that have just been mentioned are in accordance with the provisions of European directive No. 46 from 1996 (96/46/EC of 16 Jul. 1996), in particular with respect to the following characteristics:                for each of the substrates and each level:                    the mean of the recovery rates should be between 70 and 110%;            the repeatability, expressed as variation coefficient (ratio of the standard deviation to the mean for the sample concerned, expressed as a percentage) should be at most 20%;                        for each of the substrates, the total variation coefficient (all levels included) should be at most 20%.        
Another known method for analyzing fosetyl-Al is described in an article entitled Rapid determination of fosetyl-Al residues in lettuce by liquid chromatography/electrospray tandem mass spectrometry (Hernandez et al., Journal of AOAC International, Vol. 86, No. 4, 2003).
The method described concerns the quantification of fosetyl-Al residues in plant samples that are derived from lettuce. The method requires a step consisting of extraction with water by means of a high-speed mixer, followed by the injection of a 5-fold diluted extract into a liquid-phase chromatograph.
The fosetyl-Al is therefore quantified by liquid chromatography coupled to electrospray tandem mass spectrometry after addition of tetrabutylammonium acetate as an ionic pairing agent.
The analysis of samples of lettuce enriched at 2 and 0.2 mg/kg is reported. The limit of quantification is 0.2 mg/kg, whereas the limit of detection of fosetyl-Al is 0.05 mg/kg.
However, many of these known methods of analysis require a chemical derivatization step. Such an additional step complicates the analysis and very substantially prolongs the duration thereof. Furthermore, the implementation of this step requires specific expertise and increases the financial cost of these methods.
In addition, during such a derivatization step, the derivatizing agents used, which may be TMSD, diazomethane or other alternative derivatizing agents, are reactants which, in addition to their high cost, present considerable risks when they are used. Among the risks encountered when using such derivatizing agents, mention may be made of their toxicity and also their explosiveness. The use of such agents also results in a high cost.
Moreover, these known methods comprise many handling steps (evaporations, re-dissolutions, sample transfers) increasing, accordingly, the loss and dissemination of the compounds to be analyzed. Such a dissemination of compounds can also pose the problem of its environmental impact, in particular when the effluents derived from these methods of analysis are retreated.
Furthermore, these known methods have the major drawback of not being specific for particular compounds. This lack of specificity can result in compounds for which the protection and quantification characteristics are similar, not being differentiated. Other known methods have the disadvantage of only allowing the analysis of fosetyl-Al alone, without being able to carry out the simultaneous analysis of phosphorous acid, for example.
Some of the known methods are described only for particular matrices; for example, one already known method concerns only particular plant tissues derived from lettuce.
Finally, these known methods do not make it possible to achieve certain stricter limits of quantification, in particular the limits of quantification ensuing from recent regulations, for example directive 96/46/EC of 16 Jul. 1996.